On the slices of sensorimotor and frontal cortex in layer V of guinea pigs the diversity of neuronal spontaneous activity, the mechanism of its origin and functional specificity were studied. In both regions, neurons that did not have spontaneous activity predominated (39% in the sensorimotor cortex and 32% in the frontal cortex) over neurons with any other firing levels. The iontophoretic application of the excitatory transmitter, glutamate, caused activation spike reactions in all registered neurons; moreover, short-term activation reactions to glutamate had a significantly longer after-discharge in neurons of the frontal cortex (up to 2500 ms and more) compared to reactions of neurons of the sensorimotor region. This means that postexcitatory hyperpolarization in nerve cells of the frontal cortex is less expressed and, therefore, they have a lower density of K+ channels on their membranes. With an increase in the level of spontaneous activity, K+ membrane permeability decreases, which is confirmed by the appearance of a long activation reaction to acetylcholine (which blocks K+ channels), exactly when spontaneous firing appears in “silent” neurons. Despite the fact that spontaneous activity is formed by glutamatergic excitation, its considerable diversity is associated with the structural and membrane characteristics of neurons, which determine the different degrees of EPSPs attenuation on the way of moving along dendrites. Acetylcholine regulates this process in different ways, in accordance with different states of K+ membrane permeability. Therefore, the lower content of K+ channels on the membranes of neurons of the frontal cortex does not allow regulating spontaneous activity in the same range as occurs in neurons of the sensorimotor region. The presence of a high proportion of spontaneously inactive neurons in the cortex (higher in the sensorimotor cortex) suggests that cortical neurons are generally characterized by a high density of K+ channels and a significant increase in firing response to acetylcholine, while spontaneously active neurons cannot control the spontaneous activity in a wide range.